蛋白酶体功能与帕金森病相关性实验研究

The Study on the Relationship between Proteasome Function and Parkinson Disease

目的观察蛋白酶体抑制剂诱导大鼠黑质多巴胺能神经元α-突触核蛋白(-αsynuclein,-αSyn)的表达及聚集,探讨蛋白酶体功能在帕金森病(PD)发病中的作用机制。方法采用立体定向将蛋白酶体抑制剂Lactacys-tin注射至大鼠黑质部位。以免疫荧光法观察黑质区多巴胺能神经元变性缺失,并应用免疫荧光双标法观察多巴胺能神经元内蛋白聚集的包涵体及其主要成分-αSyn的表达,然后通过原位杂交分析-αSyn mRNA表达及Western印迹法检测黑质-αSyn表达量改变。结果注射Lactacystin第7天大鼠开始出现自发性活动减少,阿扑吗啡尚可诱导出旋转行为;3周后患侧黑质部位酪氨酸羟化酶(TH)阳性细胞明显减少。TH与硫磺素、硫磺素与-αSyn复合染色呈阳性。-αSyn mRNA表达量升高,蛋白表达水平增加。结论Lactacystin诱导大鼠黑质细胞-αSyn表达升高并出现蛋白聚集可能是导致PD发病的机制之一。

Objective To investigate α-synuclein expression and aggregation in dopaminergic neurons of rat substantia nigra exposed to proteasome inhibition for the role of proteasome function in the pathogenesis of Parkinson disease. Methods Lactacystin, a proteasomc inhibitor, was unilateral infused stereotaxically into the substantia nigra of rats. The loss of dopamincrgic neurons induced by Lactacystin in the substantia nigra were detected by imunofluorescence staining. The double labeling fluorescence staining was used to observe co-staining of TH and Thioflavinc,as well as Thioflavine and α-synuclein. The level of mRNA of α-synuclein was determined with in situ hybridization and expression of α-synuclcin was detected by Western blotting. Results The rats began to display spontaneous and apomorphinc induced rotation 7 days a（ter Lactacystin injection into the substantia nigra. On the 21 day after the injection of Lactacystin, the loss of TH positive neurons in substantia nigra was observed. The colocalization positive neurons of TH and Thioflavine, Thioflavine and α-synuelein was observed in the imaging of double labeling immunofluorescence. The level of mRNA of α-synuclein was high, Western blotting showed the expression α-synuelein was high as well. Conclusions Laetaeystin can induce up-regulation and aggregation of α-synuclein in the dopamincrgic neurons of substantia nigra, and proteasome maybe act as a main role in the pathogcncsis of Parkinson disease.